Development of Stable Packaging and Producer Cell Lines for the Production of AAV Vectors

Abstract

Today, recombinant adeno-associated virus (rAAV) vectors represent the vector systems which are mostly used for in vivo gene therapy for the treatment of rare and less-rare diseases. Although most of the past developments have been performed by using a transfection-based method and more than half of the authorized rAAV-based treatments are based on transfection process, the tendency is towards the use of stable inducible packaging and producer cell lines because their use is much more straightforward and leads in parallel to reduction in the overall manufacturing costs. This article presents the development of HeLa cell-based packaging/producer cell lines up to their use for large-scale rAAV vector production, the more recent development of HEK293-based packaging and producer cell lines, as well as of packaging cell lines based on the use of Sf9 cells. The production features are presented in brief (where available), including vector titer, specific productivity, and full-to-empty particle ratio.

Keywords: CAP cells; HEK293 cells; HeLa cells; Sf9 cells; adenovirus; baculovirus; herpes simplex virus; rAAV; stable inducible packaging and producer cell lines.

Figure 4

Molecular constructs used for the development of the different HEK293 cell based packaging/producer cell lines. (A) Developments performed by Qiao et al. [74]: Construction of dual-splicing switch AAV packaging plasmid; the switch system is based on the Cre-Lox system; (B) Constructs used for the development of an AAV producer cell line [78].

Figure 5

Molecular constructs used for the development of the different insect cell based packaging cell lines. (A) Constructs developed by Aslanidi et al. [50]; (B) Constructs developed by Mietzsch et al. [87] in the frame of the OneBac development (AAV1-12 rep/cap constructs used for establishment of the Sf9 packaging cells); (C) Constructs developed by Wu, Y., et al. [88] in the frame of the OneBac 2.0 development; (D) Moreno, F., et al. [89] did not modify the rep construct but developed a combined cap—rAAV construct in order to dispose of a flexible system for easily switching from one AAV serotype to another one and from one transgene construct to another one. Thus, the rep construct was similar to that used by Mietzsch et al. [87]; (E) Rep construct developed by Moreno et al. [90].

Figure 5

Molecular constructs used for the development of the different insect cell based packaging cell lines. (A) Constructs developed by Aslanidi et al. [50]; (B) Constructs developed by Mietzsch et al. [87] in the frame of the OneBac development (AAV1-12 rep/cap constructs used for establishment of the Sf9 packaging cells); (C) Constructs developed by Wu, Y., et al. [88] in the frame of the OneBac 2.0 development; (D) Moreno, F., et al. [89] did not modify the rep construct but developed a combined cap—rAAV construct in order to dispose of a flexible system for easily switching from one AAV serotype to another one and from one transgene construct to another one. Thus, the rep construct was similar to that used by Mietzsch et al. [87]; (E) Rep construct developed by Moreno et al. [90].

Figure 1

Genome organization of wild-type AAV. A scale of 100 map units is used (equivalent to about 4700 nucleotides). (Top) T-shaped dark boxes indicate the ITRs. The horizontal arrows indicate the three transcriptional promoters (P5, P19, P40). (Bottom) The solid lines indicate the transcripts, and the introns are shown by the broken lines. A poly-adenylation signal present at map position 96 is common to all transcripts. The first ORF encodes the four regulatory proteins (rep 78, rep 68, rep 52, rep 40) for which transcripts arise from the promoters P5 and P19 in combination with alternative splicing. The second ORF driven by promoter P40 encodes the three capsid proteins (VP1, VP2, VP3) from two transcripts. VP1 is initiated from the first cap transcript, and VP2 and VP3 are translated from two different start codon sites from the second cap transcript. Note that the translation initiation site of VP2 is an ACG. The second cap transcript encodes the assembly-activating protein (AAP), necessary for AAV capsid assembly (from Galibert and Merten [24]).

Figure 2

rAAV design and production principle (in a triple-transfection setting). AAV helper (rep, cap), rAAV vector, and the adenoviral helper (E2A, E4orf6, VA RNA genes) plasmids are brought into HEK293 cells (which are E1A, E1B positive) by Ca-phosphat or PEI transfection. Notes: prom—promoter; ivs—intervening sequence (e.g., intron); pA—polyA sequence.

Figure 3

Establishment of packaging and producer cell lines for rAAV vector production. The transfection of HeLa or A549 cells with a plasmid harboring the rep2 (from AAV2)-capx (from any AAV serotype) sequences (p-rep-cap in the figure) leads to the establishment of a packaging cell line. When these cells are either transfected with a pAAV-vector (plasmid) followed by infection with an helper virus (wtAd) orinfected with adenovirus (wtAd) and 24 h later with an E1-deleted adenovirus—AAV-Hybrid virus (providing the rAAV transgene sequence flanked by the two ITRs) (Ad-Hyb-AAV-vector)—rAAV as well as adenovirus are produced. If the rAAV transgene sequence is stably integrated via plasmid transfection (pAAV-transgene) or vector transduction (rAAV-vector), the packaging cells become stable producer cells. Upon infection with adenovirus (wtAd), these cells start to produce rAAV vector and adenovirus (wtAd).